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In recent years, gamma-hydroxybutyrate (GHB) usage for drugging in nightlife contexts has increased noticeably. GHB spiking refers to an instance where droplets of said drug are added to a person’s drink without them knowing [1] . This can increase their vulnerability to being robbed, sexually assaulted, and a number of other risks. GHB is the most commonly used ‘rape drug’ in Western Europe [2] . It has a fast onset of action, and can cause permanent damage in excessive doses or when mixed with alcohol [3] . Symptoms of a high dosage of GHB include apnea, heart attacks, cardiac arrest, losing of consciousness or becoming comatose, and blood pressure problems, with symptoms having become severe enough to result in death in a number of cases [4] .

To better understand the problem, we first had to identify our customers and concentrate on a specific geographical area. We focus on Western Europe, specifically the United Kingdom, Belgium, the Netherlands, and France. In these countries, the amount of suspected spiking incidents has increased significantly in recent years [5] [6] [7] , despite the well-organized healthcare systems in place. As an iGEM team based in the Netherlands, we chose to conduct our research mainly here.

A large portion of the Western European younger population says they have experience with their drink being spiked, or know someone whose drink was drugged. A European survey of sexism-free-night [5] reported that out of the around 4500 people who filled in the survey, 7.8% thought they had been drugged through “Premeditated [means] or with prior preparation", meaning someone made them purposefully consume a drug without them being aware of it. Another research showed that in the UK, one in nine women thinks their drink has been spiked at least once, and 28% of the interviewed people know someone whose drink is suspected to have been poisoned [6] . In the Netherlands, the Nederlandse Omroep Stichting (NOS) surveyed 5000 people; 5% of surveyees said they suspected to have been spiked, and over 50% of questioned women answered that they take the possibility of being drugged into account when going out [7] .

Even though the media attention and the reported incidents have increased, there is little data or scientific proof to support these claims [8] [TU Delft, interview Steven Biemans ]. Until now, GHB detection technologies have been slow, inconvenient, and expensive. In addition, due to the rapid breakdown of GHB molecules in the body, the drug is unlikely to be identified in the body six hours after consumption [9] . As a result, hospitals cannot confirm most cases of drugging, leading to a lack of data and no evidence. Consequently, the victims are not able to file a police report, and perpetrators cannot be prosecuted [TU Delft, interview Amber Schaafsma and Cornelius ].

Portable tests for GHB already exist but are expensive, single-use, and slow. Furthermore, none of the specialists we spoke to had ever heard of anyone utilizing them in a bar, nightclub, or similar setting [TU Delft, interview Amber Schaafsma, Steven Biemans and Querine Hoejenbos ]. Moreover, hospitals in the Netherlands use high-performance liquid chromatography (HPLC) to test for GHB, a procedure that requires large devices operated by professionals [TU Delft, interview Amber Schaafsma ], learn more about it in the market analysis section . This delays the detection process and lowers the efficiency of the emergency response. Fast, on-site, accurate GHB testing is required to prevent the consumption of the spiked drink, learn more about the frequency of GHB spiking, and enhance the emergency response.

Remark: needle spiking

Recently, the media has reported on a novel practice known as needle spiking [10] . The cases have not yet been verified, but experts argue that due to GHB's high viscosity and the requirement for relatively large amounts of the drug to be present in the body before symptoms present, it cannot be used as a drug for needle spiking [11] [12] . This means that if cases of needle spiking do occur it has to happen with other drugs, meaning this problem is outside of the scope of this project.

To develop the right solution to treat the problem of GHB spiking, we have to better understand the exact problems and subproblems at hand. A root-cause analysis was done based on literature research and interviews with relevant stakeholders (see Integrated Human Practices ). The ‘5x Why?’ approach was used to find the underlying causes. See Figure 1. It was found that the lack of adequate self-tests is the main underlying problem [13] .

It is easy to spike someone with GHB

We talked to various organizations related to clubs, bars, and festivals, and we found that spiking people with GHB is relatively easy. An employee working for the Trimbos-institute informed us that GHB can be purchased quite easily [TU Delft, interview Steven Biemans ]. In fact, this drug is affordable and easy to find on the market [8] . Moreover, there is a lot of information freely available online on how to produce GHB using only legal compounds [14] . Spiking requires small volumes of the drug, which are easy to smuggle inside the venue. Additionally, it might be challenging to secure your drink inside clubs, festivals, and bars, because customers are sometimes disoriented by their surroundings as a result of the busy environment and alcohol intake. Lastly, people tend to not test drinks at festivals due to the high prices and inconvenience of the tests currently on the market (see market analysis section ).

GHB cases are not reported

Various experts told us GHB is hard to detect because of the short half-life and the behavioral change of the victim when drugged. Short half-life refers to the amount of time required for a quantity to decrease to half of its starting value [15] . A drug with a brief half-life typically penetrates the blood-brain barrier quickly and can have almost immediate effects [16] . But since the molecules degrade very quickly, it is hard for a detection system to detect them 3 to 6 hours after consumption [9] . In addition, one of the symptoms of GHB contamination is aggression, which makes it very difficult for doctors or healthcare employees to test the patient [TU Delft, interview Amber Schaafsma ]. Suspected GHB incidents are not noted without valid testing, meaning no police report can be successfully filed. This leads to a severe lack of data regarding this problem, as well as a lack of justice for both the victim and perpetrator. In addition, no tests are done because existing tests are inconvenient. Most of the options on the market are expensive, single-use, slow, and require active testing, a feature that is disruptive in a nightlife scenario. See the market analysis section for further assessments.

Empathy Map

An empathy map is a tool used to analyze the thoughts, feelings, and objectives of customers to determine their needs in light of seven questions, as can be seen in Figures 2 and Figure 3. We identified two consumer segments through literature review and interaction with our stakeholders: end users (people partaking in nightlife scenarios) and hospitality businesses (see Integrated Human Practices ). We applied the empathy mapping tool to both customer segments [17] .

WHO are we empathizing with?

It is critical to analyze the consumers' identities, the circumstances they find themselves in, and their precise roles in these events. In this case, the hospitality businesses and people engaging in the nightlife, so-called users, are our target clients. The first group wants to provide a safe environment for their customers [TU Delft, interview Querine Hoejenbos, Jetti Steffers and Survey to clubs ], and the latter want to enjoy their time without thinking about the possibility of being spiked [TU Delft, Focus Group and Survey to users ]. Therefore, preventing GHB spiking is crucial in creating a secure and enjoyable setting.

What do they DO?

Users:

People engaging in nightlife activities are concerned about drink-spiking. For this reason, they implement measures to protect themselves, including covering their drinks, avoiding accepting drinks from strangers, monitoring the bartenders while they are pouring their drinks, and never leaving their drinks unattended [TU Delft, Focus Group and Survey to users ]. If they decide to buy our product, they will stop worrying about this aspect, and therefore will not have to adopt the previously mentioned measures. When our biosensor detects GHB it will emit light. When the client sees the light, they will bring the glass to the bartender to ask for another one, therefore alerting the venue about the presence of GHB and avoiding the consumption of the poisonous substance altogether.

Hospitality Businesses:

Hospitality Businesses implement in-house rules to prevent unsafe situations. In addition, some entities train their employees to prepare them in case of emergency, including in the eventuality of involuntary drug consumption. In The Netherlands, this is done in collaboration with the Trimbos Institute, an institute responsible for the research of drugs and alcohol on mental health, and Gemeentelijke Gezondheidsdienst (GGD) [18], a Dutch service center for public health [TU Delft, interview Querine Hoejenbos]. With our product, the customer will ensure a safe environment for their clients and will be able to identify the problem within minutes, leading to faster emergency management.

What do they SEE, SAY, and HEAR?

Drink-spiking, particularly GHB-spiking, has received much media attention during the past few years. The subject has been covered extensively in news stories and social media profiles, making readers more fearful [19] [4] [5] [6] [7]. Additionally, several movements that aim to give victims a voice have emerged globally [20] [21]. There is not always evidence to support these assertions. Rather the opposite is true: due to the fast degradation of the GHB molecule in the body [9], it is quite challenging to demonstrate the spiking, resulting in a massive lack of data [TU Delft, interview Steven Biemans]. Moreover, the low quality of the test available on the market increases the inability to have a clear understanding of the issue [ market analysis section ]. With our product, the lack of data will be solved, leading to a better understanding of the magnitude of the problem.

What do they THINK and FEEL?

Pains and gains are two ways that our consumer segment's feelings and opinions might be represented. The difficulties in the hospitality industry originate from the fact that visitors do not feel secure there. Users are apprehensive and constantly on edge when participating in nightlife activities because of the widespread conjecture around this issue [TU Delft, Focus Group and Survey to users ]. This is demonstrated by all the precautions they take when engaging in the night scene. With our device, the nightlife will be more enjoyable for consumers as they will not have to worry about GHB spikes, which will also benefit hospitality firms.

Market Analysis

By conducting a market analysis [22] and consulting several experts, we found that there are two primary categories of tests currently available on the market: 1) preventative and 2) detection tests. The distinction lies in the fact that for the first category of tests, the user does not consume the substance because they are prevented from doing so. For the second category, the test is used to identify the presence of GHB in the victim’s body.

Detection systems

The GHB detection technologies that are already available on the market can be divided into tests carried out at healthcare facilities, and in-person tests [23] [24] [25] [26] [27] [28] that can be purchased online and performed at nightclubs and other social gatherings by the customer themselves.

Hospitals do not usually screen for GHB, as we learned during an interview with first aid physician Amber Schaafsma [TU Delft, interview Amber Schaafsma]. This is because GHB is not detected by the typical urine drug test, which tests for the presence of cocaine, cannabis, amphetamines, and opiates [29]. A trained pharmacist must perform High-Performance Liquid Chromatography (HLPC) to identify GHB specifically, which takes several hours and is thus inconvenient in an emergency case [TU Delft, interview Amber Schaafsma]. When a victim is thought to have been poisoned by GHB, they are treated according to the alcohol poisoning procedure. While this is an inaccurate diagnosis, the treatment is effective for the patient [TU Delft, interview Amber Schaafsma]. Since patients are not tested for GHB due to the inconvenience of the method, the victims cannot successfully file a report because they are left without any evidence of the accident.

To provide a thorough picture of the market scenario for on-site test techniques, we made a table (see Table 1) comparing the three most important characteristics a GHB test should have. We listed the GHB tests according to cost, detection time, and ease of use. By all means, the most crucial qualities are accuracy and precision, meaning a minimal amount of false negatives and false positives, but all the tests mentioned have adequate values for these parameters. Furthermore, with the exemption of HPLC, all tests are single-use.

Prevention systems

Additionally, some products stop GHB from being added to drinks in the first place. Drink covers prevent anything from being added to the drink [30], while specific coasters react when in contact with GHB [31]. The covers have the drawback of requiring the use of a straw, which is undesirable for some beverages, such as beer. Moreover, some of our interviewees believed some bartenders spike drinks with GHB before placing the cover on the glass, making them ineffective or even giving customers a false sense of security. The coasters instead are not very practical in absence of a table.

Table 1. Market Analysis of detection methods for GHB

Test	Cost	Detection Time	Type of Test	Ease of Use	Availability
ALLTEST GHB Urine Drug Test Kit Cassettes [23]	$6.57	Within 10 minutes	Urine test with buffer	Medium	Not available
Sip Chip [24]	$14.99 for 5 single-use test	Less than 30 seconds but does not detect GHB	Paper Assay	Medium	Not available
Detectachem [25]	$24.90 for 10 counts	2/3 minutes	Swab Sticks	Low/Medium	Available
TestSure [26]	$9.99	1/2 minutes	Color Reaction	Low	Available
Test Kit Plus [27]	$20.00 for 10 single-use tests	seconds but does not detect GBL	Color Reaction	Low	Available
EZTest [28]	$3.52	seconds	Color Reaction	Low	Available

Overall, it is clear that a faster, cheaper, and more reliable test is needed on the market. Moreover, it is crucial to try and develop a test that is passive, as all the methods listed above require active testing, a practice that is inconvenient in the nightlife setting.

Patients must be tested for GHB in both private and public healthcare facilities. Due to the need for remote laboratories and qualified personnel, the current testing procedures are time-consuming, expensive, and difficult to access. The GHB concentration in drinks may be measured quantitatively, modularly, easily, and affordably with our solution, SPYKE. Thanks to our test, customers will be able to get results in only a few minutes.

The differences between regular glass and SPYKE are shown in Figure 4, while the mechanism of our transcription factor-based capacitive biosensor is displayed in the video. Our cup is composed of three layer, one containing the beverages, one hosting the electrode, and the last one holds the arduino. SPYKE employs the transcription factor's, BlcR, ability to bind and unbind DNA in presence of GHB. The dissociation caused by GHB generates a change in capacitance which activates the system. As seen in Figure 4, when the system is operational, an orange light will turn on. For more information check the design and the hardware page.

The electrical system is advantageous because:

• Since GHB is already present in some drinks at a low concentration, it is essential to set a clear threshold. The electrical system is the greatest choice for this, and therefore prevent false positives.
• The reaction time is usually faster compared to a biological based output.

The three-layered glass is advantageous because:

• SPYKE is considered a food-contact material (see Safety). Due to the fact that the Arduino is not in contact with the beverage, the three layers allow us to give the maximum level of safety.
• SPYKE places a high focus on recycling. This technology makes it feasible to reuse electronic components more than once, resulting in a less environmental imprint.

The dissociation between the transcription factor and the DNA is advantageous because:

• The molecules needed for the biosensor are easy to produce.
• The sensor's quick reaction time gives our customer the option of quick testing.

The market for biosensor SPYKE contains two customer groups: hospitality businesses and people that engage in the nightlife [TU Delft, section Empathy Map]. The businesses are responsible for providing a safe environment for the second group. They do this by implementing house rules and by educating their employees. Querine Hoejenbos, an employee from the Koninklijke Horeca Nederland (KNH), explained that this is done through a collaboration between the Trimbos Institute and Gemeentelijke Gezondheidsdienst (GGD) regarding safety measures and drug consumption management [TU Delft, interview Querine Hoejenbos].

SPYKE creates value for these customers through its:

High quality

The quality of our product is the aspect most highly valued by both parties. Therefore, in the manufacturing of SPYKE, emphasis is placed on quality assurance. In practice, this means that our product will be manufactured in Europe because the European Union has high-quality standards that are recognized globally. In addition, our company will be involved in every step of the process to perform quality checks.

Superior functionality

The hospitality businesses and the people that engage in the nightlife are in need of a GHB detection method that is safe, specific, and fast-acting [see local context ]. Current detection and prevention tests are accurate but require active testing, are inconvenient, and are not fast enough [see market analysis section ]. Therefore, the users are not able to enjoy their night safely without actively taking countermeasures [TU Delft, Focus Group and Survey to Users]. SPYKE represents a unique combination of safety and speed that would relieve the customers of this dilemma.

Point of care

The current methods to obtain data on GHB spiking include methods like High-Performance liquid chromatography (HPLC). To conduct these analytical methods, laboratories and trained pharmacists are required, as explained by Amber Schaafsma [TU Delft, interview Amber Schaafsma]. This method is inefficient since it requires a lot of time [see market analysis section ]. In addition, this does not prevent the spiking, but it only checks for the molecule's presence in a victim’s body once they have already been drugged. Our product allows hospitals to perform the test at the facility without outsourcing samples to a professional in a laboratory, which is both easier for all parties involved and enables a better treatment to commence earlier.

Affordability

To make the test accessible to all potential end-users, a low price is required. Through affordable systems, we have a price that is cheaper than similar tests currently on the market. More information about the current market scenario can be found in the market analysis section [see market analysis section ], while our prices can be found in the business model canvas [see business canvas model ].

Radical innovativeness

Besides a unique functionality, SPYKE represents a radical innovation in the field of biosensors [TU Delft, section patenting]. The concept of a capacitive biosensor triggered by transcription factor dissociation is novel and innovative. Because of the novelty and applicability, we also patented our idea.

We evaluated SPYKE's drawbacks and benefits. A sensitivity analysis was used to find potential hazards first. The second step was the development of strategies to minimize these risks. We then compared the advantages and disadvantages. A larger overview of the risks and mitigation plan is discussed in the safe-by-design section.

Sensitivity Analysis

In this sensitivity analysis, the following risks are identified as potential negative outcomes of the implementation of SPYKE:

• Increase fear of GHB spiking

• Increase in GHB production

• Increase in use of other drugs like ketamine and benzodiazepines for spiking

• Increase in needle spiking

• Food safety legislation

• Non-proper handling

• Barrier to technological diffusion

• Bad protection of users

By consulting stakeholders (see Human Practices ), we were able to categorize these risks according to their likelihood and severity using a scale of 1 to 5, with 5 being the most severe, as is shown in Figure 5. The risk mitigation plan should prioritize the risks with a higher score. The explanation of the position of each risk within the matrix is described below.

Secondary effects and consequences of a GHB biosensor

The existence of a GHB biosensor could cause a lot of secondary effects leading to:

Increase fear of GHB spiking

SPYKE might bring more attention to the problem of GHB spiking which could lead to an increase in fear regarding this problem.

• Probability (1), as the media already heavily covered the subject, and the awareness is already high.

• Impact (2), as an increase in fear could result in more paranoia in the nightlife, but this would mostly lead to more protection of drinks which does not have a large negative impact.

Increase in GHB production

SPYKE provides information regarding GHB and its production in the local context and root-cause analysis . This could lead to an increase in GHB production.

• Probability (1), as there is already a lot of information available on GHB production on the internet.

• Impact (3), as the increased production could lead to more GHB addiction and spiking.

Increase in use of other drugs like ketamine and benzodiazepines for spiking

SPYKE would bring a solution to GHB spiking specifically, which could push people to replace GHB with other drugs like Rohypnol and ketamine.

• Probability (3), as Leanneard Borra explained there are other suitable options for spiking next to GHB.

• Impact (2), as these drugs are more easily detectable than GHB and easier to dose.

Increase in needle spiking

Needle spiking is the practice of spiking someone by injecting the drug directly into the victim with a needle or syringe. Since drugging through drink spiking would become more difficult with the implementation of SPYKE, perpetrators could opt for needle spiking to spike their victims instead.

• Probability (1), as the experts we talked to agree that the chance of needle spiking becoming a large problem is small.

• Impact (4), as it is very hard to protect yourself from needle spiking.

The analysis of the probability and the impact are explained in integrated human practices .

Food safety legislation

A sensor containing oligonucleotides and a protein purified from a GMO as a food contact material may not be easily allowed into the market due to food safety issues.

• Probability (1), as we talked to Dutch and European food safety institutions and they agreed the sensor could pass the legislation.

• Impact (5), as without SPYKE the situation would remain the same, and no similar systems could be developed for other purposes.

Non-proper handling

There is a risk that SPYKE is not properly handled by either businesses or end-users. For example, the output could be misread, the device could be poorly cleaned or badly assembled at the location. This would result in a worse or malfunctioning sensor.

• Probability (3), as doctors told us people often have difficulty using or reading out quick tests.

• Impact (4), as a percentage of the tests might work less accurately resulting in distrust in SPYKE and possibly biosensors in general.

Barrier to technological diffusion

End-users may be discouraged to use SPYKE as they lack the knowledge and awareness of the technology.

• Probability (1), as when SPYKE works better than competitors it will likely be easily accepted. Also, SPYKE contains no living organism which also increases the acceptability.

• Impact (2), as people will not use SPYKE to be protected from GHB spiking, which also means the lack of data is not resolved.

Bad protection of users

SPYKE could detect GHB poorly or inaccurately, resulting in problems caused by having an unnoticeable output signal, or showing many false positives and negatives.

• Probability (2), as SPYKE uses a novel system which means there could be as-of-yet unknown difficulties. However, the electrical component of the system counteracts this by enabling one to make many small changes to the system easily, making it more flexible and adaptable to unforeseen circumstances.

• Impact (4), as the sensor might work less precisely than expected meaning its readings may not be trustworthy.

Mitigation plan

Secondary effects and consequences of a GHB biosensor

• Increased fear of GHB spiking
• Increase in GHB production
• Increase in use of other drugs for spiking
• Increase in needle spiking

For all of these points, regular research should be done to find the actual influence SPYKE may have. Sensors for other rape drugs can be combined together with our GHB sensor to prevent drugging with other substances. In safe-by-design this is expanded upon.

Food safety legislation

To make sure SPYKE passes the food safety requirements, many design changes were made to meet the requirements [ integrated human practices ]. Close contact should be kept with relevant institutions to quickly adapt the sensor if problems arise.

Non-proper handling

To make sure SPYKE is handled well, a QR code will be placed on the bottom linking to user manuals for both the end users and responsible nightlife facilities. It may also be opted for to gather all SKYPE cups to a central location after use to ensure they function properly for the next user.

Barrier to technological diffusion

To increase knowledge and awareness of our sensor, details on the use and the possible benefits should be transparent through open science practices and a straightforward website. Moreover, the education of people on the safety of our sensors will be one of our main branding points.

Bad protection of users

As we work with an electrical system, changes can be made very easily. Constant data on the workings of our sensor should be gathered and surveys on customer satisfaction should be conducted to ensure the system works optimally.

Risk-benefit balance

• Rapid: A drawback of current methods is the time required to perform the test. Our sensor uses a protein unbinding mechanism working on the order of seconds or quicker, compared to the tests on the market which take dozens of minutes. Providing a quick result is important when checking drinks for potential spiking.
• Passive: Our sensor measures GHB constantly in your drink. Tests available on the market require active testing, which means every time you want to test your drink you have to perform an action. As our sensor needs no attention, this provides a large advantage over existing tests.
• Multi-use: The tests on the market are all single-use. This means every time you want to test your drink you have to use a new test. Our test can be used continuously throughout the night and most parts can be recycled or directly reused.
• Accurate: Accurate: After talking to hospitals, the forensic institute, and the Netherlands Public prosecution service we found out the current GHB quick tests are not accurate [see integrated human practices ]. Due to the electric system's ability to handle data and correct for certain factors, our test will both be more accurate and more precise.

Conclusion

SPYKE has many advantages over the competition, but there are risks associated with the legislation, use, and recycling of the system. By making design changes, providing relevant information, and continuously monitoring the performance of SPYKE, these risks can be minimized. We can confidently state that SPYKE’s benefits outweigh the possible risks.

Patenting

We learned that when a product or idea is novel, innovative, and industrially applicable (and that this can be verified), the creator can apply for a patent. This information was obtained via the patenting handbook of the 2020 Leiden iGEM team [32] and during our interactions with V.O. Patents & Trademarks. The biosensor created by SPYKE integrates a transcription factor in a capacitive sensor, which makes it a pioneer in the field of biosensors. Therefore, if there is a desire to commercialize SPYKE, we were recommended to submit a patent application. We approached Blijke Kroezen, a patent attorney at V.O. Patent and Trademarks, and, after recognizing the potential of our project, she suggested that we pursue a priority patent. The patent was filed on 30/09/2022 thanks to her help.

It covers the method including the protein and DNA sequences, and the hardware that we used. It also mentions our application: a GHB biosensor.

This priority application offers several advantages. First, it gives us a year from the filing date to develop the idea further and demonstrate its viability before submitting a more expensive international patent application (PCT). The filing date, or "priority date", is registered in the meantime. This means that we will still be able to maintain exclusivity on our technique even if someone reveals a comparable discovery after this time. Therefore, a priority patent application gives us the opportunity to create intellectual property (IP) with less financial outlay.

Second, filing for a patent makes it simpler to draw in investors and persuade partners to work with us. Our technology may easily be used by a bigger opponent with more resources if we didn't get a patent for it [TU Delft, section Porter’s Five Force Analysis ]. This would significantly lower SPYKE's value to investors. By safeguarding our intellectual property, we own the technology. This increases the company's appeal to financiers and business associates, making it simpler to get funding and other resources [33].

Manufacturing

Manufacturing may start after it has been established that there is a market for a biosensor of SPYKE's specifications and that it can be made cost-effectively and reliably to the level of quality required to meet the market criteria. When making plans to produce a biosensor, safety and packaging are the two primary factors to consider [34].

When production shifts to a large-scale operation, safety becomes a top priority. When handling the product, certain safety precautions must be taken into account as the sensor contains biological elements, including DNA strands and proteins. Additionally, SPYKE requires the use of GMOs during its manufacturing, thus safety measures must take this into account as well. It is also essential to have protocols in place to be able to safely dispose of components containing biological elements. Measures are also necessary for the electronics component. For instance, alteration of the electrode during manufacturing calls for solvents that, when used in large quantities, could pose a health risk; this is a factor that needs to be considered. Overall, an efficient ventilation system and air conditioning system should be adopted in areas where electronic components are prepared, which could significantly affect the budget of the company [TU Delft, section Cost plan ].

Another crucial component of manufacturing is packaging. A sealed container would be preferable because biosensors are sensitive to their surroundings. Since the product needs to be food-safe, we will comply with the current rules and use recycled plastic, rPET, as the main material both for the cup and its packaging.

As was already indicated, GMOs are necessary to lower SPYKE's long-term costs. Therefore, the European manufacturer of SPYKE is chosen to assure the safety of the production process, our manufacturing staff, and the test itself. We have chosen to locate our business in the Netherlands because we are more familiar with both the situation regarding drink spiking and regulations concerning working with GMOs here. Thanks to our stakeholders we already have relationships with suppliers, potential clients, and sponsors [TU Delft, section integrated human practices ].

Distribution

In a perfect world, we would utilize the current transportation network to distribute SPYKE. The best choice would be to freeze dry the product because it contains biological components that must be preserved for an indefinite period of time. Since the issue of requiring a low temperature would no longer exist, this would also benefit transportation.

Furthermore, it is our responsibility to abide by Dutch legislation as our company will be situated in the Netherlands. The Dutch Civil Code is based on contractual freedom, which states that distributors and suppliers are only held to the agreements they have made directly with one another. There are, however, a few requirements for distribution agreements. Most of these are derived from competition rules [35].

Compliance

To launch SPYKE on the market, our test and read-out device must comply with local laws and regulations. Therefore, SPYKE should abide by regulations that are upheld by the Netherlands, the European Union (EU), and the United States of America (USA). To get approval from all the regulatory bodies, certifications must be acquired that guarantee the safety of the different components of our product. An overview of the most pressing regulations and certifications can be found in Table 2.

Table 2. Overview of main certifications and regulations that apply to SPYKE.

Certfication	Regulatory body	Type
FDA approval [36]	U.S. Food & Drug Administration FDA	Food, drugs, and medical devices
CE Marking [37, 38]	European Economic Area (EEA)	Safety, health, and environmental protection
FCC certification [37, 39]	U.S. Federal Communications Commission	Electromagnetic emissions
IEC 62133 [37, 40]	International Electrotechnical Commission (IEC)	Lithium-free batteries
RoHS [37, 41]	European Parliament (EU)	Lead-free products
FCM certification [42]	European Food Safety Authority	Food Contact Material
Regulation (EC) No 1935/2004 [43]	European Food Safety Authority	Food Contact Material

Project Plan

The Gantt chart in Figure 6 shows the anticipated length of time needed for the SPYKE rollout. Three categories were taken into account for this: the research and development stage, the legal admission, and the development of the company itself.

Research and Development stage

We will put a lot of effort into the research and development of our biological and electrical components. In the first scenario, our main focus will be on tailoring the DNA oligo and the protein to increase the binding affinity [TU Delft, sections Module 2 and Module 3 ], which is crucial for a quick response.

Optimization is also necessary for the reaction conditions. The concentrations of DNA and proteins have an impact on how quickly the sensor reacts. We market our product as fast-acting, hence the biosensor must be able to identify GHB in a matter of minutes if not quicker. Additionally, because the item is within a glass, it will constantly interact with the beverage, which is a highly variable environment. Since the temperature [44] and pH [45] in this condition can change significantly over time and between different contents, our biomolecules must be adaptable to this shift. A solid development plan and in-depth research will be needed for this.

Consequently, we will concentrate mostly on the electronics to shrink the size of our sensor and validate our hardware. Additionally, we will assess its response to external stimuli since it will be exposed to the same environment as the biological component.

Legal Admission

The company will be registered during the first month of 2023. Since we have already filed a priority patent, we will have to proceed and work on the main claims we filed to confirm them for the international patent. Moreover, we will need to apply for a food safety certificate, which is required for any product in contact with food or beverages [42].

Company

For our product to be commercialized, funding is needed. Investors’ interest determines the length of time needed for fundraising. Agreements with suppliers and distributors of the test components are necessary, in addition to funds. As soon as the research and development phase is completed, negotiations with suppliers and distributors may begin since by that time, we will know how the biosensor will look when all the parts are in place. Following these negotiations, the real manufacture, packaging, and rollout begin. We plan to launch the product in the first quarter of 2025 officially.

Porter’s five forces analysis

Porter’s five forces analysis [46] outlines elements that can assist companies in comprehending the advantages and disadvantages of a specific market or business. Porter's 5 forces are (1) Competition in the industry, (2) Potential of new entrants into the industry, (3) Power of suppliers, (4) Power of customers, and (5) Threat of substitute products [46].

Rivalry among existing competitors

The market for rapid, reliable, passive, and on-site tests is restricted nowadays. All the tests available on the market require active testing, as presented in the market analysis section. Moreover, the test methods for GHB in-situ are very expensive and single-use, characteristics that we know are not favored by the customers [TU Delft, survey to users ]. Nowadays, almost no one tests GHB in their drink in clubs or bars even though there is a general concern regarding this topic, which leads us to suspect that there isn’t a valuable alternative to our product. In hospitals, GHB is tested through a laborious procedure that requires specific laboratory equipment and trained pharmacists. This results in a slow protocol and inefficient first aid response [TU Delft, market analysis ]. Due to its greater accessibility, lower cost, modularity, and lack of a need for active testing, SPYKE sets itself apart from both detection and prevention tests. This makes us believe that once we enter this market, the disparity in products between SPYKE and its rivals will not be disadvantageous to our business.

Threat of new entrants

The risk of new entrants into our potential market heavily depends on the obstacles to entering this sector. The following is a list of constraints that keeps rivals out of the Western European GHB biosensor market. The threat of new entrants into the business is minimal as a result of these entry restrictions [47].

1. Legal barriers to entry

Our exclusive right to make the SPYKE technology is protected by a patent. As a result, it is against the law for competitors to copy our developed technique. Additionally, particular permits are needed in order to test this product. Companies must get a specific institutional permit in order to work with GHB because it is classified as a drug in List 1 of the Dutch Opium Act [48]. This permit can take up to a year to secure, which stifles potential competitors from entering this market.

2. Technical barriers to entry

In order for industries to enter this market, they need solid initial capital due to the high-startup costs. Laboratory equipment is very expensive, as well as the biological and electrical supplies needed to develop such a product. Additionally, there are a lot of sunk expenses in this sector, including those for marketing, research, advertising, distribution, and development. When developing the budget for the new company, these expenses, which are not always recoverable, must be considered.

Moreover, a significant amount of knowledge in both the biological and electrical disciplines is needed for this kind of undertaking. Due to the expertise required that must be met before entering the industry, this factor reduces the number of possible competitors.

3. Strategy barriers to entry

Due to the fact that the product we provide has not yet reached the market [TU Delft, market analysis ], SPYKE qualifies as a "first mover". With this title, we have a strong brand identity and excellent visibility. As a result, rivals would have to spend a significant amount of money on branding and getting their industry acknowledged. Being the first to market would also help us build brand loyalty, which would again provide a challenge for the potential rival.

Power of buyers

Customers of SPYKE are classified as individual users and public hospitality businesses [TU Delft, empathy map ]. We strive for a test that is affordable and available to everyone, regardless of socioeconomic status, to lessen reliance on charitable giving or health insurance. As a result, we must maintain a reasonable price for our product [TU Delft, cost analysis ]. Additionally, we want businesses to buy numerous tests in a single order from a single facility [TU Delft, proposed implementation ]. The purchasers' negotiating strength is increased by these two requirements. However, the bargaining power of clients is diminished because SPYKE has a special test that is not yet offered on the market [TU Delft, market analysis ]. As a result, the industry is only moderately impacted by consumer bargaining power.

Threat of substitute products or services

A potential substitute product could be a device that tests for multiple drugs, and not only GHB. This would provide a multi-use product that could further improve the user’s safety. The aforementioned entrance obstacles must be surmounted in order for a corporation to create and use a multi-detection biosensor that satisfies the demands for sensitivity, specificity, accuracy, and quality. Furthermore, it is difficult to find biosensors on the market that can test for numerous compounds at once, necessitating additional study and a greater level of competence.

Power of suppliers

Providers are required to provide SPYKE with the following four key resources:

• Biological components of the sensor [TU Delft, parts ]
• Substrate for GMOs that produce cell-free systems and plasmids [TU Delft, parts ]
• Membrane filters for the electrical system [TU Delft, proposed implementation ]
• Electronics for the hardware [TU Delft, hardware ]
• Plastic cup with the components descriped in hardware [TU Delft, hardware ]
• O rings to improve the watertightness[TU Delft, hardware ]

Regarding the first two criteria, we are using a short DNA strand and a particular protein, which makes us a minor customer in this market because macromolecules like these are frequently used in laboratories. Large business fields also frequently use filters, which guarantees us a lower incidence of their revenue. We use a minimal circuit for the hardware's electronics in order to provide the safest end result to our clients, which implies that the costs are too low to compete with those of tech giants.

PESTEL analysis

The design of SPYKE and its suggested implementation were based on discussions with stakeholders . These specific decisions may be influenced by political, economic, social, technological, legal, and environmental issues. A PESTEL analysis was done to determine which decisions have a high likelihood of being impacted by these factors.

SWOT analysis

The so-called SWOT matrix can be used to condense SPYKE's strengths, weaknesses, opportunities, and threats after defining all internal operations and performing an external company study. While opportunities and threats represent the external business environment and its benefits and drawbacks, strengths and weaknesses describe the advantages and downsides of the internal operations of the firm. Figure 9 displays this SWOT analysis.

Responsible Innovation

We place high importance on sustainability from an environmental and socioeconomic standpoint. Here, we go over our plans for implementing these values in our company. Our solution should guarantee long-term sustainability in order to responsibly address the issue of drink spiking with GHB.

Socio-economic sustainability

SPYKE must be advantageous to those involved in any phase of its life cycle in order to be socio-economically viable, or at the very least have minimal adverse effects. We plan to accomplish this by:

Monitoring unintended side-effects

Since our method is innovative, there is a chance that it could have unintended negative impacts, like those listed in the risk and benefit analysis. We intend to increase the socio-economic sustainability of our sensor by keeping an eye on such adverse effects through the analysis of the test data and by testing our device’s performance.

Not exerting monopoly power

We are the unique provider of SPYKE thanks to patenting. If SPYKE users become dependent on it, the monopoly position could be abused by requesting greater profits. Promoting fair competition, partnerships, public oversight, and anti-monopoly laws and regulations should avoid this.

Environmental sustainability

We aim to reduce the ecological footprint of each stage of SPYKE's life cycle from the perspective of environmental sustainability. To do this, we performed a Life Cycle Analysis on these phases and developed methods for reducing our ecological footprint.

Raw material extraction

In order to create SPYKE, raw resources like gold, amino acids, nucleotides, and copper are required. We should build a connection with suppliers who place an equal emphasis on environmental sustainability, such as through the bio-production of gold and the use of renewable energy, in order to reduce the environmental impact of extracting these raw materials. To minimize transportation, it is preferable that these suppliers be located close to the manufacturing location.

Manufacturing and processing

SPYKE’s manufacturing process was created by us. This method uses heat exchange and continuous batch scheduling to save energy expenses. We would make an effort to use as much renewable energy as possible throughout the manufacturing of the biosensor.

Transportation

SPYKE will make use of the pre-existing distribution network already in place across Europe. To cut down on transportation expenses and carbon emissions, we established our company in the Netherlands [see manufacturing ].

Waste

SPYKE’s implementation is built on a system of recycling [see proposed implementation ]. We want to ensure the quality of the sensors by collecting used cups, and recycling and recharging components in-house only. This is done to minimize the risk of sensors becoming defective due to improper handling by others. To achieve this, a strong line of contact between the hospitality industry and our firm needs to be established. The majority of the components of our biosensor can be recycled or reused thanks to SPYKE's design [see integrated human practices ].

Our goal is to create an on-site deployable GHB passive biosensor. We hope to increase data availability on GHB drink spiking and lower its incidence by creating and putting the test into practice. To assess SPYKE's potential, a business model was developed. The business model canvas template is used to describe our company plan.

There are nine different areas in the business model canvas. The core ideas of our product are at the center of the business model. The customer groups, client relationships, distribution channels, and income streams are used to characterize the sales on the right side of the canvas. The important partners, key activities, key resources, and the cost structure on the left reflect the internal operations.

Cost structure and revenue streams

The production of the test, ongoing research and development, transportation, and other unstructured activities are the sources of SPYKE's expenses. These costs need to be offset by the income streams generated by SPYKE in order to break even and eventually turn a profit. The manufacturing costs can be divided into two categories: costs of the biological parts and costs of the hardware. By considering the two primary elements of the test—the oligonucleotide and the transcription factor, BlcR—an evaluation of the expenses of the biological components was made. Table 3 provides a breakdown of the pricing of each of these parts per cup. Table 4 lists the prices of the hardware prototype per cup, which are described in detail in the hardware manual.

Table 3. Costs of the biological part.

Component	Cost
Oligonucleotide (IDT)	€1,14
Plasmid pET11a-BlcR	€1,78
Total	€2,92

Table 4. Costs of the hardware.

Component	Cost
Electronics	€47,12
3D printing	€10
Other	€4,41
Total	€61,53

Transport and packaging are two additional expenses that are directly tied to the product. The revenue streams SPYKE generates from the sale of the product must be used to pay for these expenses. Start-ups are typically encouraged to estimate margins of 50% to 60% of the total costs of the product [49]. This buffer was raised to 100% of the previously calculated cost of the biological parts and additional equipment because it is difficult to determine the exact expenses associated with these activities in the early stages of research and development (R&D) (present stage of SPYKE). Since the expenses listed here are for the proof-of-concept prototype and we anticipate a significant reduction in the costs for the manufacture of the final hardware, 50% margins were decided for the hardware after comparison.

Funds need to be raised in order to pay for the costs incurred during the early start-up period. This can be accomplished by attracting investors, as well as by pursuing funding and innovation competition applications. The product and the business need to be pitched to potential investors in order to acquire the former. The company model and the market potential are covered in the pitch. Together with Delft Enterprises and SkylineXD, we collected input on our business approach. We looked at funding options as well and discovered that there are sizable research grants that we could apply for from organizations like the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO) [50].